Screening of Heat-Resistant Morchella Strains and Elucidation of Their Heat-Tolerance Mechanisms

Simple Summary

Morchella, a prized edible and medicinal fungus, faces growing cultivation challenges due to rising global temperatures. Identifying heat-tolerant strains and elucidating their adaptive mechanisms are therefore crucial for sustaining production. In this study, we screened 19 Morchella strains under heat stress and identified HLM as highly thermotolerant and JY as heat-sensitive. Physiological and transcriptomic analyses revealed that HLM maintains cellular homeostasis through enhanced antioxidant activity under heat stress.

Abstract

Morchella is a nutritious and artificially cultivable rare ascomycete, and its growth and development regulation mechanisms are a current research hotspot. High-temperature stress severely limits the annual yield of Morchella, and this challenge is intensifying with global warming. However, previous studies have lacked systematic screening for heat-tolerant Morchella strains, and their molecular response mechanisms to heat stress remain unclear. In this study, we conducted a comprehensive analysis of phenotypic characteristics, physiological metabolism, and transcriptomics on 19 Morchella strains under normal (25 °C) and high-temperature (30 °C) conditions. The heat-tolerant strain HLM exhibited superior performance in mycelial growth, morphology, and field cultivation. It maintained cell homeostasis under heat stress through mild osmotic regulation (elevated levels of proline, soluble sugars, and proteins), a robust antioxidant system (increased activities of CAT, POD, and SOD), and reduced malondialdehyde accumulation. Transcriptomic analysis identified a novel regulatory model of “stress perception—metabolic preparation—terminal detoxification” in the heat-tolerant strain HLM under heat stress. The rapid upregulation of the SMPD1 gene may mediate ceramide signal generation, promoting G6PDH expression to drive carbon flow into the pentose phosphate pathway, thereby increasing NADPH output. As the detoxification terminal, AKR4C uses this reducing power to eliminate toxic carbonyl end products like malondialdehyde, completing the defense loop. These findings offer new insights into the heat-tolerance mechanisms of large ascomycetes, provide a theoretical foundation for stress-resistant Morchella breeding and cultivation in high-temperature areas, and serve as valuable resources for exploring heat-tolerance mechanisms and molecular breeding in other edible fungi.

1. Introduction

Morchella, a member of the Morchellaceae family [1], is one of the few artificially cultivable edible fungi in the phylum Ascomycota. Its notable culinary and medicinal values have made it highly sought after in international markets [2,3]. Cultivation of Morchella was achieved in China in 2012, and the industry expanded from an initial 200 hectares to 16,466 hectares by 2022, driven by low labor requirements, short initial investment periods, long production cycles, and high profitability [4]. In just 10 years, yields increased more than 80-fold, and cultivation spread rapidly to over 20 provinces nationwide [4]. However, as the industry has developed, problems have emerged. Morchella is a typical low-temperature edible fungus and is highly sensitive to environmental temperature [5,6]. Compared with the past, global warming has caused a gradual rise in average temperatures and a marked increase in the frequency of extreme high-temperature events [7,8]. During artificial cultivation, even brief high-temperature stress can reduce Morchella yield or cause complete crop failure [6]. In the context of global warming, breeding heat-tolerant Morchella strains and developing methods to sustain and maximize yield have become urgent priorities.

Research on edible fungi under high-temperature stress has long attracted attention, particularly given climate change’s effects on agriculture. Temperature is a key environmental factor controlling edible fungi growth and development. During cultivation, high-temperature stress causes accumulation of organic acids produced by hyphae [9], significantly inhibits hyphal growth rate [10], destabilizes cell membranes, and triggers accumulation of reactive oxygen species (ROS) and disruption of metabolic pathways [11,12]. The impact of heat shock on edible fungus cultivation has become increasingly severe [13]. Current research on heat stress in edible fungi mainly focuses on Basidiomycota. Phenotypic studies show that prolonged high temperature elongates the stipe and thins the pileus of Lentinula edodes, substantially reducing yield [14,15]. High temperatures above 23 °C slow development and reduce the size of Agaricus bisporus fruiting bodies, impairing A. bisporus quality and yield [16]. Heat stress also darkens the ectomycorrhizal fungus Tuber borchii and weakens its ability to persist on host roots [17]. At the molecular level, endogenous NO in Pleurotus ostreatus hyphae under heat stress reduced oxidative damage by inhibiting aconitase (ACO) gene and protein expression [18,19]. Trehalose induced by reactive oxygen species (ROS) during heat shock was also crucial for P. ostreatus survival [20]. In Ganoderma lucidum, heat shock regulates mycelial growth through a soluble Ca²⁺-mediated signaling pathway [21]. Additionally, a 42 °C heat treatment for 2 h significantly increases the polysaccharide content in Ganoderma lucidum fruiting bodies [22]. In Agaricus bisporus, exogenous PABA addition or transgenic overexpression of PABA synthase from strain 02 enhances the heat tolerance of the heat-sensitive strain 8213 [23]. In Pleurotus giganteus, significant upregulation of heat shock proteins, DnaJ proteins, and zinc finger proteins was identified as a key response pathway under 40 °C high-temperature stress [24]. However, research on the adaptive heat shock mechanisms in large ascomycetes remains scarce, with only a few reports, such as the effect of heat shock on the metabolites of Cordyceps militaris [25]. In Morchella, the understanding of the systemic molecular regulatory network underlying its thermotolerance response remains fragmented and inadequate. Existing research, integrating transcriptomic and proteomic analyses to decipher the molecular mechanisms of heat shock in Morchella, has revealed that thermotolerant strains specifically upregulate the ubiquitin ligase Rsp5, which in turn promotes the expression of heat shock proteins (HSPs) [5]. Furthermore, studies have demonstrated that heat stress significantly impacts the yield, physiological traits, and morphological characteristics of Morchella [26]. Evaluating heat tolerance through morphological indicators, mycelial yield, and physiological characteristics is crucial. Establishing a mycelial heat tolerance identification system can aid in exploring high-quality strains suited to local resources and climate. This provides a theoretical and technical foundation for local introduction and high-yield cultivation, promoting the sustainable and healthy development of the local Morchella industry.

By identifying individuals that exhibit the greatest phenotypic divergence under identical stress conditions, this method maximizes the enrichment of key genetic and molecular signals directly associated with the target trait. This allows for clearer revelation of core regulatory pathways amidst complex biological background “noise”. Consequently, this study utilized 19 Morchella strains (including 11 M. sextelata, 7 M. eximia, and 1 M. importuna strains) as materials. Through high-temperature stress treatment, combined with analysis of mycelial growth morphology, field cultivation phenotype, and physiological metabolic characteristics, a strain (HLM) with excellent comprehensive thermotolerance and stable phenotype was screened. We further assessed the heat stress adaptability of HLM strains through biomass determination and fluorescein diacetate (FDA) fluorescence staining. Additionally, we employed transcriptomics to compare gene expression differences between the extreme heat-tolerant HLM strain and the extreme heat-sensitive JY strain. This comparison aimed to identify key genes and pathways involved in the heat tolerance of Morchella, thereby providing preliminary insights into their molecular regulatory mechanisms. Our findings enhance the understanding of fungal heat stress responses and offer a foundation for future molecular breeding of Morchella and the development of heat-resistant strains in other edible fungi.

2. Materials and Methods

2.1. Sample Collection and Heat Shock Treatment

The 19 strains of Morchella were utilized in this experiment, sourced from the Edible Fungi Center at Northwest A&F University. These included 11 strains of M. sextelata, 7 strains of M. eximia, and 1 strain of M. importuna; all are commercial strains cultivation in China. There is no consensus on the optimal temperature for Morchella cultivation [27]. However, Morchella often face temperatures exceeding 25 °C in field conditions, which leads to a decline in both growth rate and biomass of morel mycelium when temperatures surpass 25 °C [28]. In this experiment, 25 °C served as the control, while 30 °C was used to simulate sublethal heat stress. This temperature effectively induces typical heat-stress phenotypes in Morchella mycelia and facilitates the differentiation of thermotolerance among different strains. The 19 strains were inoculated onto Potato Dextrose Agar (PDA) and incubated in the dark at 25 °C for 7 days. Agar blocks from the mycelial tips were then transferred to fresh PDA medium. Separate control and heat stress groups were established, with five biological replicates for each strain. The control group was kept in the dark at 25 °C for 8 days. The heat stress group was incubated at 25 °C in the dark for 4 days, followed by 30 °C in the dark for another 4 days. Additionally, sterile cellophane was placed on the culture medium in the same batch of experiments. Once the mycelium covered the medium, it was collected, placed in cryotubes, rapidly frozen in liquid nitrogen, and stored at −80 °C for future use.

2.2. Determination of Mycelial Growth Rate and Observation of Mycelial Morphology

The germination time and growth rate of each mycelial strain were recorded. The mycelial growth rate was measured using the cross-line method along two perpendicular axes. Measurements were recorded at 24 h intervals, and the average growth rate was calculated for both the control and heat stress groups. Throughout cultivation, observations and records were made of the growth conditions and morphological characteristics of the mycelium in each group. Adobe Photoshop software was used to analyze the color of the mycelium in the photographs. The scores obtained from this analysis were then used to calculate rankings via the membership function method. Uij = (Xij − Xjmin)/(Xjmax − Xjmin). Uij represents the membership function value of the j indicator of the i strain; Xij represents the measured value of the j indicator of the i strain; Xjmin represents the minimum value of the j indicator; Xjmax represents the maximum value of the j indicator. Since the same scale (1–3) and the same normalization formula were applied uniformly to all traits, each trait contributed with equal weight to the subsequent comprehensive evaluation. Detailed protocols for the assay are provided in Supplementary File S1.

2.3. Field Cultivation Validation

Based on the cultivation method summarized by Liu et al. (2017) [29], we further standardized the Morchella cultivation management protocol (Supplementary File S2). The cultivation trial was conducted in a solar greenhouse. Within the greenhouse, the land was divided into several small plots, with each plot planted with one variety. Three replicate controls were established for each, ensuring consistent climatic conditions and soil physicochemical properties during cultivation for different varieties. Yield data were obtained by measuring the total fresh weight of all fruiting bodies produced within each small plot of different varieties over the entire cultivation cycle.

2.4. Determination of Physiological and Metabolic Characteristic Indicators

Begin by taking 0.5 g of frozen mycelium and grinding it into a powder using liquid nitrogen in a mortar. Add 5 mL of pre-cooled phosphate buffer (PBS, 0.1 M, pH 7.4) to the powder. Allow the mixture to stand on ice for 30 min, ensuring even mixing by vortexing every 10 min. Centrifuge the mixture at 4 °C and 12,000 rpm for 20 min. The resulting supernatant is the crude enzyme solution. Malondialdehyde (MDA) content is determined using the thiobarbituric acid method; soluble sugar (SS) content is measured using the anthrone colorimetric method; soluble protein (SP) content is assessed using the Coomassie brilliant blue method; proline (Pro) content is determined by the acidic ninhydrin method. The contents of catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) are measured using a commercial assay kit (Beijing Boxbio Science & Technology Co., Ltd., Beijing, China), following the manufacturer’s instructions. Detailed protocols for all assays are provided in Supplementary File S3.

2.5. Biomass Measurement and FDA Fluorescence Staining Validation

We selected high-temperature tolerant strains (HLM) and high-temperature sensitive strains (JY) for the study. Agar blocks from the mycelium tips were inoculated into a liquid medium. The control group was incubated at 25 °C and 120 rpm for 9 days. In contrast, the heat stress group was incubated at 25 °C and 120 rpm for 7 days, followed by 2 days at 30 °C and 120 rpm. Each group included three biological replicates. Mycelial pellets were collected, centrifuged at 5000 rpm for 10 min, and the supernatant was discarded. The mycelial pellets were washed and precipitated with distilled water three times, then dried and weighed. The samples were then transferred to an 80 °C oven and dried for 48 h to constant weight. The high-temperature inhibition rate was calculated using the formula: [(1 − stress group biomass/control group biomass) × 100%].

To verify FDA fluorescence staining, begin by taking mycelial pellets and washing them three times with PBS. Add 1 mL of FDA working solution and incubate the mixture in the dark at 25 °C with shaking at 50 RPM for 20 min. Next, introduce 10 μL of PI working solution and continue incubating in the dark for an additional 5 min. Centrifuge the mixture at 5000 RPM for 5 min and discard the supernatant. Wash the pellet three times with PBS, then resuspend it in 500 μL of PBS. Take a 10 μL sample, place it on a slide, and observe under a fluorescence microscope.

2.6. Transcriptome Sequencing Analysis and Real-Time Fluorescence Quantitative PCR Validation

RNA extraction, cDNA library construction, and sequencing were conducted as follows: total RNA from Morchella mycelium was extracted using the TRIzol kit(Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). RNA concentration was measured with a MD microplate reader (Molecular Devices, San Jose, CA, USA), and RNA integrity was assessed using a Qsep400 bioanalyzer (BiOptic Inc., Taiwan, China). The cDNA library was constructed following the kit’s instructions. Upon completing the library construction, initial quantification was performed with a Qubit 2.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). The Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) was then used to determine the library’s insert size. Once the insert size met expectations, RT-qPCR was employed to accurately quantify the library’s effective concentration, ensuring it exceeded 2 nm for quality assurance. After confirming library quality, sequencing was executed using the PE150 strategy on the Illumina platform. The raw data underwent filtering, sequencing error rate assessment, and GC content distribution analysis using fastp [30]. Subsequently, clean reads were aligned to the reference genome with HISAT (2.2.0) to obtain positional information on the reference genome and sequence characteristics specific to the samples [31].

Genomic reads are aligned and assembled into transcripts using StringTie act (2.2.2). Subsequently, based on comparison results and gene locations on the reference genome, the read count for each gene is determined. Gene function annotation for unigenes was performed using seven major databases (Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KEGG, GO). The read count data from the gene expression analysis are standardized using DESeq2R (1.16.1) software. Significantly differentially expressed genes (DEGs) between samples were screened using thresholds of padj < 0.05 and |log2FoldChange| > 1.

The experimental design comprised four groups, each with three biological replicates: the heat-resistant strain control group (HLM-control), the heat-resistant strain high-temperature treatment group (HLM-heat), the heat-sensitive strain control group (JY-control), and the heat-sensitive strain high-temperature treatment group (JY-heat). DEGs were identified by comparing samples between different groups using DESeq2 1.48.0, conducting four sets of differential analyses: HLM-control vs. HLM-heat, JY-control vs. JY-heat, JY-control vs. HLM-control, and JY-heat vs. HLM-heat. GO and KEGG functional enrichment analyses were performed on the DEGs to identify the main metabolic and signal transduction pathways involved. Some DEGs related to lipid metabolism and energy metabolism under heat stress were selected for real-time fluorescence quantitative PCR (qRT-PCR) validation. Total RNA from different Morchella RNA samples was reverse transcribed into first-strand cDNA using the HiScript III RT SuperMix for qPCR kit (Vazyme Biotech Co., Ltd. Nanjing, China). Act1 was used as the internal reference gene. Primers were designed with Premier 5.0 (Supplementary File S4). RT-qPCR reactions followed the ChamQTM Universal SYBR qPCR Master Mix Kit (Novozyme Biotechnology Co., Ltd., Nanjing, China) instructions. Gene expression levels were analyzed using the relative quantitative 2^−ΔΔCt method.

3. Results

3.1. Mycelial Growth Rate and Morphological Phenotypes of Different Morchella Strains Under Heat Shock Conditions

The 19 Morchella strains were subjected to heat stress at the mycelial stage. The results showed that the growth rates of strains G4, HLM, 118, MGQM, and XL1 under heat stress showed no significant difference (NS) compared to the control, indicating stronger heat tolerance (Figure 1A). The mycelial growth rates of the other 14 strains decreased significantly under heat stress (Figure 1A). Analysis of the reduction ratio in growth rate after stress revealed that strains ZGX, JY, K9, 301, and CMQM exhibited the greatest reduction, all exceeding 35% (Figure 1B), indicating their higher sensitivity to heat stress.

The growth status and morphological characteristics of the 19 Morchella strains under heat stress were scored. The obtained scores were ranked using the subordinate function method for analysis (

Table 1. Analysis of the morphological characteristics of mycelia ^$.

Strain Number	Germination Time	Hyphal Growth Vigor	Hyphal Color	Hyphal Density	Edge Regularity	Growth Rate (cm/h)	Average Value of Membership Function	Comprehensive Ranking
HLM	1	3	3	3	3	0.045	0.97	1
401	1	3	3	3	3	0.040	0.93	2
7-5	1	3	3	3	3	0.032	0.85	3
301	1	3	3	3	3	0.030	0.83	4
MGQM	1.5	3	2	3	3	0.042	0.81	5
118	1.5	3	2	2	3	0.043	0.74	6
CMQM	1.5	3	2	3	3	0.031	0.72	7
XL1	1.5	2	2	2	3	0.048	0.71	8
G4	1.5	2	2	2	2	0.042	0.56	9
GL4	1.5	2	2	2	2	0.039	0.54	10
LM11	1.5	2	2	1	2	0.044	0.50	11
T3	2	2	2	1	2	0.040	0.42	12
7-6	2	2	1	1	2	0.042	0.36	13
LXQM	2	2	1	1	1	0.040	0.26	14
ZDT	2	2	1	1	1	0.038	0.24	15
JY	3	1	1	1	1	0.039	0.08	16
K9	2.5	1	1	1	1	0.031	0.06	17
GYH	3	1	1	1	1	0.035	0.05	18
ZGX	3	1	1	1	1	0.034	0.04	19

^$ Germination Time (Scores 1–3): “1” indicates the shortest time and strongest growth activity; “1.5” indicates moderate time and good growth activity; “2” indicates longer time and moderate growth activity; “2.5” indicates relatively long time and weak growth activity; “3” indicates the longest time and weakest growth activity. Hyphal Growth Vigor (Scores 1–3): “3” indicates vigorous growth and robust hyphae; “2” indicates moderate growth and stable growth state; “1” indicates weak growth and poor growth state. Hyphal Color (Scores 1–3): “3” indicates hyphal color is white and uniform; “2” indicates hyphal color is relatively white; “1” indicates hyphal color is grayish-white. Hyphal Density (Scores 1–3): “3” indicates hyphae are dense and cover large areas; “2” indicates hyphal density is moderate and cover area is appropriate; “1” indicates hyphae are sparse and cover small areas. Edge Regularity (Scores 1–3): “3” indicates hyphal edges are neat and regular; “2” indicates hyphal edges are relatively neat; “1” indicates hyphal edges are irregular.

). Strains HLM, 401, 7-5, and 301 had higher comprehensive scores, indicating vigorous growth, uniform and healthy mycelial color, dense growth, and regular colony margins. In contrast, strains ZGX, GYH, K9, and JY had lower scores, indicating weak growth vitality, uneven color and poorer health status, sparse growth, and irregular margins. Comprehensive analysis showed that strain HLM performed excellently in both growth rate and heat tolerance, ranking first overall, making it suitable for further promotion and application. Strains ZGX, JY, and K9 performed poorly in both growth rate and heat tolerance.

3.2. Phenotypic Evaluation of Agronomic Traits in Field Cultivation for Different Morchella Strains

To evaluate the agronomic performance of 19 Morchella strains, a cultivation trial was carried out in a simple solar greenhouse. The fruiting cycle, yield, and overall field performance of each strain were detailedly recorded and evaluated. Most strains exhibited a fruiting cycle of approximately 80 days. Notably, strains JY and T3 exhibited shorter fruiting cycles of 78 and 77 days, respectively. This suggests their rapid mycelial growth at normal temperatures, which is consistent with their vigorous performance during the mycelial stage. Observations were made at 10, 30, and 60 days post-sowing to assess mycelial growth initiation, mycelium bloom, and primordia distribution density. Systematic observation and data analysis revealed significant growth differences among strains. By day 10, strains ZDT, GL4, JY, T3, K9, HLM, and CMQM demonstrated superior mycelial development, with higher mycelial density and healthier colony morphology compared to other varieties. By day 30, the mycelial networks of strains ZDT, GYH, JY, T3, G4, and HLM had matured, achieving optimal mycelial bundle diameter and branch density. By day 60, strains ZDT, GYH, JY, T3, and HLM entered the reproductive growth stage, exhibiting high primordial differentiation ability. In contrast, strains ZGX, 118, 301, and LXQM remained in the vegetative growth stage with no primordia formation detected (

Table 2. Field cultivation performance of different strains ^$.

Strain Number	Mycelial Density on 10 and 30 Days After Wowing		Primordium Density on 60 Days After Sowing
	10 Days	30 Days
T	+++	+++	***
GL4	+++	++	**
ZGX	+	++	-
GYH	++	+++	***
7-6	++	++	*
LM11	++	++	**
JY	+++	+++	***
T3	+++	+++	***
K9	+++	++	**
G4	++	+++	**
HLM	+++	+++	***
7-5	++	++	*
118	++	++	-
401	++	++	**
301	+	+	-
LXQM	++	++	-
MGQM	++	++	**
XL1	++	++	**
CMQM	+++	+	*

^$ Mycelial density: Graded as “+++” (very dense, covering large areas), “++” (moderately dense, covering moderate areas), and “+” (sparse, covering small areas). Primordium density: Graded as “***” (high density, numerous and clustered), “**” (medium density, numerous and evenly distributed), “*” (low density, few in number), and “-“ (undetected).

).

All 19 Morchella strains in this experiment successfully completed reproductive growth, forming complete fruiting bodies. Morphogenesis indicators are detailed in

Table 3. Agronomic traits of different strains.

Strain Number	Fresh Weight of Individual Mushrooms/g	Fruit Length /mm	Pileus Length /mm	Pileus Diameter /mm	Stipe Diameter /mm	Yield/(kg/m2)
ZDT	23.81 ± 2.91 ab	103.12 ± 1.05 a	71.56 ± 2.15 bc	35.78 ± 1.89 abc	26.12 ± 1.67 ef	1.00 ± 0.17 de
GL4	22.72 ± 2.12 b	102.45 ± 1.67 ab	70.23 ± 3.45 bcd	34.89 ± 2.67 bcd	25.56 ± 2.34 fg	0.79 ± 0.18 cd
ZGX	15.05 ± 5.94 f	100.12 ± 3.12 bc	65.00 ± 5.00 fgh	29.85 ± 5.00 j	27.12 ± 2.67 cde	1.06 ± 0.18 cd
GYH	22.14 ± 3.75 b	99.24 ± 1.98 bc	69.78 ± 1.98 cd	34.56 ± 1.98 cd	25.23 ± 5.01 gh	0.95 ± 0.09 de
7-6	17.96 ± 5.22 e	94.78 ± 4.23 d	65.56 ± 3.21 f	31.89 ± 3.21	29.12 ± 2.15 ab	1.16 ± 0.22 bc
LM11	19.76 ± 4.12 d	98.52 ± 3.45 c	67.34 ± 3.78 def	33.12 ± 3.78 ef	23.78 ± 4.89 k	0.86 ± 0.15 e
JY	20.33 ± 3.44 c	97.89 ± 4.89 c	68.45 ± 4.12 d	33.78 ± 4.12 de	24.56 ± 6.78 ij	1.25 ± 0.25 ab
T3	21.52 ± 3.22 c	99.87 ± 2.34	69.12 ± 3.12 cd	34.12 ± 3.12 cde	24.89 ± 3.21 hi	0.82 ± 0.07 e
K9	24.02 ± 1.53 a	104.67 ± 1.89 a	72.12 ± 1.89 ab	36.12 ± 2.15 ab	26.45 ± 6.12 def	1.08 ± 0.13 cd
G4	20.96 ± 1.83 c	97.34 ± 4.45 c	67.89 ± 2.34 de	33.45 ± 2.34 def	24.12 ± 2.56	1.24 ± 0.28 ab
HLM	24.32 ± 2.51 a	105.24 ± 1.56 a	72.85 ± 1.24 a	36.85 ± 1.24 a	26.78 ± 3.78 de	1.34 ± 0.30 a
7-5	16.81 ± 5.87 f	95.13 ± 4.12 cd	65.12 ± 4.45 fg	31.12 ± 4.45 i	28.23 ± 4.12 bc	0.99 ± 0.10 de
118	16.32 ± 5.57 f	92.35 ± 1.24	65.05 ± 1.89 fgh	30.78 ± 1.89 ij	27.89 ± 1.98 bcd	1.14 ± 0.20 bc
401	19.46 ± 4.82 d	98.45 ± 3.89 c	66.78 ± 1.67 ef	32.89 ± 1.67 efg	23.45 ± 1.05 kl	0.91 ± 0.05 e
301	15.62 ± 5.38 f	91.23 ± 2.89 d	65.02 ± 3.67 fdh	30.34 ± 3.67 j	27.56 ± 5.89 cd	0.81 ± 0.08 e
LXQM	17.27 ± 6.05 e	93.45 ± 3.78 d	65.34 ± 1.05 f	31.56 ± 1.05 hi	28.78 ± 3.45 abc	0.99 ± 0.11 de
MGQM	18.56 ± 1.22 e	93.45 ± 3.78 d	65.89 ± 2.56 f	32.23 ± 2.56 fgh	29.85 ± 1.24 a	1.31 ± 0.29 a
XL1	18.16 ± 4.51 e	96.34 ± 2.34 cd	66.23 ± 4.89 ef	32.56 ± 4.89 fg	22.85 ± 7.00 l	0.96 ± 0.14 de
CMQM	23.53 ± 2.34 ab	101.78 ± 2.67 b	70.89 ± 2.67 bcd	35.23 ± 3.25 bc	25.89 ± 4.45 efg	0.69 ± 0.03 f

Note: Different lowercase letters in the same column (or row) indicate significant differences among groups based on one-way ANOVA followed by Tukey’s HSD post-hoc test (p < 0.05).

. The data show that the biomass of individual mushrooms ranges from 15.05 to 24.32 g. The fruiting bodies’ longitudinal growth spans 91.23 to 105.24 mm, with pileus axial growth between 65.00 and 72.85 mm. Transversely, pileus spread measures 29.85 to 36.85 mm, and stipe diameters range from 22.85 to 26.78 mm. According to the NY/T 4344-2023 standard for commercial Morchella (Supplementary File S5), pileus with anaxial length of 70–110 mm are classified as large (L), while those 50–70 mm are medium (M). All tested strains meet the medium and large specifications. Notably, HLM strains exhibit superior agronomic traits. Statistical analysis of fruiting body yields revealed that MGQM and HLM strains performed exceptionally well. Additionally, the HLM, 7-5, and G4 strains possess excellent quality and appearance, making them ideal for cultivation as superior morel strains with promising returns.

3.3. Effects of Heat Stress on Osmotic Adjustment Substances in Mycelia of Different Morchella Strains

Osmotic adjustment substances, including proline, soluble sugars, and soluble proteins, enhance strain stress resistance by regulating intracellular osmotic pressure. This regulation helps maintain cellular water balance and structural stability under high-temperature stress [32]. Similarly to plants, the proline content in fungi, to some extent, reflects their stress resistance.

Strains with strong stress resistance typically accumulate more proline [33]. Determination of proline content showed that its content increased insignificantly in 7 strains: GYH, JY, K9, 118, 301, XL1, and CMQM. In MGQM, the proline content decreased under heat stress (Figure 2A), indicating weaker heat tolerance in these strains. Conversely, strains such as ZDT, T3, HLM, 7-5, and 401 exhibited relatively high proline levels after heat stress, with the most significant increases (Figure 2A,B), indicating stronger heat stress tolerance.

Higher soluble sugar and soluble protein content in fungi under stress indicate a lower possibility of cell dehydration and a higher chance of survival under adversity, signifying stronger resistance [34,35]. Determination of soluble sugar content showed that in 3 strains—ZDT, K9, and XL1—the soluble sugar content in mycelia did not change significantly under heat stress conditions (Figure 2C). The soluble sugar content decreased substantially in strains JY and MGQM after heat stress, indicating their sensitivity to heat stress (Figure 2D). Notably, the HLM and LM11 strains maintained relatively high soluble sugar levels, with the most significant increase observed, suggesting strong heat resistance (Figure 2D).

Determination of soluble protein content showed that in strains ZDT and XL1 under heat stress conditions, the soluble protein content did not change significantly. Strains JY and 118 showed a small increase in soluble protein content, while GYH showed a decrease (Figure 2E), indicating lower heat stress tolerance in these 5 strains. Conversely, 6 strains—GL4, K9, HLM, 7-5, 401, and 301—showed a higher increase and larger change amplitude in soluble sugar content in mycelia after heat stress (Figure 2E,F), suggesting they may possess stronger heat tolerance potential.

3.4. Effects of Heat Stress on Membrane Lipid Peroxidation Products and Antioxidant Enzymes in Mycelia of Different Morchella Strains

Malondialdehyde (MDA) content reflects the degree of cell membrane lipid peroxidation. Higher MDA content indicates a higher degree of membrane lipid peroxidation and more severe damage to the cell membrane [33,36]. Therefore, MDA content is usually negatively correlated with stress resistance. The research results showed that MDA content showed a significant increasing trend in most strains under heat stress. However, MDA content did not change significantly in 3 strains: HLM, 118, and XL1 (Figure 3A). In strains 7-5, 401, 301, MGQM, and CMQM, MDA content increased after stress but remained at a relatively low overall level, indicating that HLM, 118, XL1, 7-5, 401, 301, MGQM, and CMQM suffered less cell membrane damage under heat stress and thus may possess stronger heat tolerance. Strain JY had the highest MDA content in mycelia under heat stress and showed a large increase (Figure 3A,B), indicating the highest degree of membrane lipid peroxidation, the most severe cell membrane damage, and the weakest resistance to heat stress in this strain.

Catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) are key enzymes in the antioxidant system, crucial for eliminating reactive oxygen species (ROS) in the body. Consequently, they serve as important physiological indicators of stress resistance [36,37]. Typically, antioxidant enzyme activity in fungi increases under stress, with higher activity indicating enhanced ROS clearance and greater stress resistance [26,38]. Determination of CAT activity showed that under heat stress, the CAT enzyme activities of ZGX, G4, HLM, and 118 strains were relatively high. Notably, the HLM strain exhibited significantly higher CAT activity than the others (Figure 4A,B), suggesting a strong capacity to clear ROS hydrogen peroxide under heat stress. Conversely, the CAT content in T3, K9, MGQM, and XL1 strains decreased, indicating a weaker ROS clearance ability under the same conditions (Figure 4A).

Determination of POD activity showed that under heat stress, the POD enzyme activity in the MGQM strain remained relatively stable, whereas other strains exhibited a significant increase in activity (Figure 4C). Notably, strains such as HLM, T3, 401, LM11, and 7-6 demonstrated higher POD enzyme activities, with a marked increase following heat stress (Figure 4C,D). This suggests these strains possess strong antioxidant capabilities and enhanced stress resistance.

Determination of SOD activity showed that under heat stress, the SOD enzyme activity in the mycelium of ZDT, GYH, 7-6, JY, K9, and CMQM strains did not show a significant increase. In contrast, other strains exhibited a notable rise in SOD activity (Figure 4E). Notably, the 7-5, HLM, and G4 strains demonstrated the highest SOD enzyme activity, with substantial increases following heat stress (Figure 4E,F). This suggests that these strains possess strong antioxidant capabilities and enhanced stress resistance.

Based on the comprehensive analysis of mycelial growth rate, morphological phenotype, field cultivation agronomic traits, and physiological metabolic indicators under heat stress, heat-sensitive and heat-tolerant strains were screened. Strain JY performed poorly in all heat tolerance indicators and was identified as the heat-sensitive strain. Strain HLM outperformed other strains in all indicators under heat stress, indicating strong resistance to heat stress, and was identified as the heat-tolerant strain.

3.5. Growth Characteristics and Cell Viability Validation of Heat-Tolerant Strain HLM and Heat-Sensitive Strain JY Under Heat Stress

Heat-sensitive strain JY under heat stress. In this section, we verified the growth characteristics and cell activity of the heat-resistant strain HLM and the heat-sensitive strain JY when subjected to heat stress. The experiment aimed to assess the strains’ responses to elevated temperatures, focusing on their growth rates and cellular activities. The heat-resistant strain HLM demonstrated a significantly higher growth rate compared to the heat-sensitive strain JY under identical heat stress conditions. This indicates that HLM possesses inherent mechanisms that confer resistance to thermal stress, allowing it to maintain cellular functions more effectively than JY. Conversely, JY exhibited reduced growth and diminished cell activity, highlighting its vulnerability to heat. These findings underscore the importance of strain selection in environments subject to temperature fluctuations, as the ability to withstand heat stress can significantly impact overall cellular performance and viability.

Under heat stress conditions at 30 °C, significant differences emerged in mycelial growth characteristics and biomass between the heat-tolerant HLM strain and the heat-sensitive JY strain, indicating their varying adaptability to high temperatures (Figure 5A). HLM demonstrated strong mycelial growth stability, with only a 10–20% reduction in colony diameter. Its mycelial growth edges remained neat, and neither initial growth activity nor the growth process was significantly disrupted by high temperatures. In contrast, JY’s mycelial growth was notably inhibited, with colony diameters shrinking by 40%, and the mycelium exhibited a grayish-white appearance. Biomass analysis further confirmed these findings. Under high-temperature stress, HLM experienced a biomass inhibition rate of just 10%, significantly lower than JY’s 51.2% inhibition rate (Figure 5B). In summary, HLM maintained high mycelial growth vitality and biomass accumulation under heat stress, showcasing excellent heat resistance, whereas JY demonstrated inadequate resistance to heat stress.

FDA fluorescence probe detection indicated significant differences in cell survival and membrane integrity between the heat-resistant strain HLM and the heat-sensitive strain JY under both normal and heat stress conditions (Figure 5C). FDA is hydrolyzed into fluorescein by intracellular lactase in living cells, emitting green fluorescence. Dead cells or those with damaged membranes exhibit weak or no fluorescence. At normal temperatures, HLM strain mycelium forms a dense, regular network with abundant branches and intact morphology. Most mycelium is stained by FDA, displaying uniform and bright green fluorescence, indicative of healthy growth. Even under heat stress, HLM maintains high cell membrane integrity and survival rates. In contrast, under heat stress, JY strain mycelium appears sparse, with significantly reduced branches, some of which are broken or atrophied. The FDA staining shows weak and uneven green fluorescence, with only certain mycelial areas exhibiting faint signals. This suggests a marked decrease in metabolic activity, reduced cell survival, and severe membrane damage. Moreover, as shown in Figure 5D, under high-temperature stress, HLM mycelia exhibited significantly higher accumulation of osmo-protectants (proline and soluble sugars) and antioxidant enzyme activities (CAT, SOD) compared to JY mycelia, indicating a more robust stress response mechanism. Conversely, JY mycelia accumulated higher levels of malondialdehyde (MDA), suggesting greater oxidative damage. These findings highlight the superior heat tolerance of HLM compared to JY.

3.6. Analysis of Morchella Heat Tolerance Mechanism Based on Transcriptomics and Functional Validation of Key Genes

3.6.1. Quality Analysis of Sequencing Results

After high-throughput sequencing on the Illumina platform, approximately 120 million raw sequence reads (Raw data) were obtained. Low-quality reads were removed using a Phred quality score threshold (Q30 ≥ 80%), and abnormal sequences containing unknown bases (N base proportion > 5%) were simultaneously filtered out. After strict quality filtering, the total Clean reads stabilized in the range of 50–70 million. The Q30 for each sample group was above 96.55% (Supplementary File S6). The gene expression level (log₂(FPKM)) distribution for the four treatment groups ranged from −2 to 10, covering a wide range from low to highly expressed genes, reflecting good sequencing depth and gene expression detection capability. This indicates that the sequencing data quality was good and met the requirements for analysis (Supplementary Figure S1).

3.6.2. Screening of Differentially Expressed Genes (DEGs) Between Heat-Tolerant Strain HLM and Heat-Sensitive Strain JY from Transcriptome Data

To investigate the genes associated with heat tolerance in the heat-tolerant strain HLM and the heat-sensitive strain JY, we screened differentially expressed genes from transcriptome sequencing. The analysis revealed significant differences in gene expression levels, highlighting specific genes that may contribute to heat tolerance in strain HLM. These findings provide insights into the molecular mechanisms underlying heat tolerance and offer potential targets for further research and genetic improvement. In the heat-resistant strain HLM, high-temperature stress resulted in 208 differentially expressed genes compared to the control group. Of these, 124 genes were upregulated and 84 were downregulated. In the heat-sensitive strain JY, high-temperature stress led to the detection of 186 differentially expressed genes, with 75 upregulated and 111 downregulated. When comparing both strains under heat stress, 258 differentially expressed genes were identified, with 179 upregulated and 79 downregulated (Figure 6A). Heat map analysis revealed that some genes in both HLM and JY strains exhibited similar trends in expression under heat stress, suggesting these may be fundamental genes involved in the heat stress response (Figure 6B). A Venn diagram of the differentially expressed genes showed that only one gene was common across all three treatments (Figure 6C).

3.6.3. GO Enrichment Analysis of DEGs in Heat-Tolerant Strain HLM and Heat-Sensitive Strain JY

DEGs from the two varieties after heat stress were annotated and analyzed according to three major categories: biological process (BP), molecular function (MF), and cellular component (CC). The GO enrichment analysis results for DEGs showed that the GO enrichment profile for the heat-sensitive strain JY after heat stress (Figure 7B) showed a very similar trend to the GO enrichment results for JY vs. HLM under non-stress conditions (Figure 7C), but presented a completely different trend from the GO enrichment profile for the heat-tolerant strain HLM after heat stress (Figure 7A). This may be due to differences in the expression of key genes and response patterns between HLM and JY strains under heat stress, providing molecular evidence at the transcriptome level for HLM’s heat tolerance. DEGs in the heat-tolerant strain HLM under heat stress were mainly enriched in functions related to transporter activity. DEGs in the heat-sensitive strain JY under heat stress were mainly enriched in functions related to catalytic activity. Under both control and heat stress conditions (Figure 7C,D), the differences between HLM and JY strains were evident. HLM strains had higher expression levels of growth-related genes, reflecting their stronger growth potential, while JY strains had weaker basal stress defense capabilities.

3.6.4. KEGG Enrichment Analysis of DEGs in Heat-Tolerant Strain HLM and Heat-Sensitive Strain JY

The KEGG enrichment analysis of differentially expressed genes between HLM-control vs. HLM-heat and JY-control vs. JY-heat identified the top 20 pathways with the highest enrichment degrees, which were visualized using scatter plots. The analysis revealed significant differences between the HLM and JY strains following heat stress. In the HLM strain, KEGG pathways were primarily enriched in ABC transporters, biotin metabolism, fatty acid biosynthesis, and fatty acid metabolism, all of which were upregulated (Figure 8A). Notably, the upregulation of numerous transport protein genes likely contributes to the HLM strain’s robust heat tolerance. Conversely, in the JY strain, the glutathione metabolism pathway was most significantly affected, showing downregulation after heat stress (Figure 8B). This suggests that the JY strain’s antioxidant system was notably inhibited under heat stress, as evidenced by a relatively small increase in antioxidant enzyme activity, which likely contributes to its poor heat tolerance. Under control conditions, the HLM strain exhibited a higher enrichment degree in ribosome and RNA transport pathways, indicating stronger growth potential (Figure 8C). In contrast, the JY strain demonstrated lower basal metabolic activity. Under heat stress, the differences between the HLM and JY strains became more pronounced (Figure 8D), with the HLM strain displaying a more efficient protein protection and repair mechanism.

3.7. Mining of Key Genes in Heat Stress Response in Heat-Tolerant Strain HLM and Heat-Sensitive Strain JY

Transcriptome data annotation analysis revealed significant mobilization of ten single genes under high-temperature stress across various group comparisons (HLM-control vs. HLM-heat, JY-control vs. JY-heat, JY-control vs. HLM-control, JY-heat vs. HLM-heat) (

Table 4. Differential gene expression and annotation of JY and HLM under normal temperature and heat stress conditions.

Gene	Gene Function	log2FC
		JY-Control vs. HLM-Control	HLM-Control vs. HLM-Heat	JY-Control vs. JY-Heat	JY-Heat vs. HLM-Heat
H6S33-012945	Pyruvate decarboxylase (PDC)	—	1.84	1.2	—
H6S33-009348	Glucose-6-phosphate dehydrogenase (G6PDH)	−2.61	1.78	—	—
H6S33-004438	glutathione S-transferase (GST)	—	1.23	−1.30	2.47
H6S33-000202	Catalase (CAT)	−1.06	2.38	1.98	—
H6S33-012722	4-hydroxyphenylpyruvate dioxygenase (HPPD)	−2.22	1.18	−2.20	1.17
H6S33-002269	Peroxidase (POD)	—	2.09	1.01	—
H6S33-005748	Aldo-Keto Reductase (AKR4C)	−1.69	1.06	−1.59	—
H6S33-005769	Heat shock protein HSP20	−1.64	1.33	−1.94	1.62
H6S33-003407	Heat shock protein HSP70	−2.89	1.51	−1.36	—
H6S33-007274	Sphingomyelinase (SMPD1)	—	1.35	—	1.53

). Notably, genes encoding GST, HPPD, AKR4C, and heat shock proteins HSP20 and HSP70 exhibited significant expression differences. In the HLM-control vs. HLM-heat comparison, these genes showed upregulation with log2FC values of 1.23, 1.18, 1.06, 1.33, and 1.51, respectively. Conversely, in the JY-control vs. JY-heat comparison, the same genes were downregulated, with log2FC values of 1.30, 2.20, 1.59, 1.94, and 1.36, respectively. The findings indicate that these genes play a role in the regulatory mechanism of high-temperature stress in morel mushrooms. Under such stress, genes encoding heat shock proteins, enzymes, and sugars initiate stress responses to mitigate heat damage. Heat-sensitive strains exhibit less resistance to heat stress compared to heat-resistant strains.

3.8. Validation of Transcriptome Sequencing Expression Patterns by Real-Time Fluorescence Quantitative PCR (qRT-PCR)

To confirm the accuracy of the transcriptome data, the expression of 10 selected genes was analyzed by fluorescence quantitative PCR. The results showed a strong correlation with the expression trends observed in the transcriptome sequencing data (Figure 9). In the heat-tolerant strain HLM, genes encoding PDC, G6PDH, GST, CAT, HPPD, POD, AKR4C, HSP20, HSP70, and SMPD1 were upregulated. Conversely, in heat-sensitive strains, PDC, CAT, POD, and SMPD1 were upregulated, while GST, HPPD, HSP20, AKR4C, and HSP70 were downregulated. HSP20 and HSP70 are crucial for protein folding under oxidative stress, high pressure, and heat shock conditions [38]. Additionally, the AKR family complements and synergizes with the HSP system to degrade reactive oxygen species (ROS) [39]. Our findings indicated that GST, HPPD, AKR4C, HSP20, and HSP70 genes were upregulated in heat-tolerant strains but downregulated in heat-sensitive strains. Furthermore, PDC, CAT, and POD genes showed greater upregulation in heat-tolerant strains. This suggests that heat-sensitive strains have a reduced capacity to eliminate ROS and mitigate oxidative damage compared to heat-tolerant strains.

4. Discussion

Morchella is an edible and medicinal fungus [40]. In recent years, with the maturation of cultivation techniques, the field cultivation of Morchella has expanded rapidly [41]. However, Morchella can only be cultivated according to seasonal temperature changes and may experience high-temperature weather in the following spring. These high temperatures reduce mycelium metabolic activity, hinder growth, and cause stress damage. To develop strains with greater resistance to heat stress, we employed transcriptomics to analyze heat-sensitive and heat-insensitive strains, aiming to better understand Morchella response mechanisms to high temperatures.

High temperature inhibits fungal growth [10], and different types or varieties of edible fungi exhibit varying sensitivities to temperature. In this study, through preliminary screening experiments on the collected 19 Morchella strains, we found that strain HLM outperformed strain JY in terms of mycelial growth rate phenotype and physiological metabolic characteristics under heat stress (Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5). Therefore, we determined that strain HLM is insensitive to high temperature, while strain JY is sensitive. Experiments on biomass and cell activity (FDA) supported this conclusion (Figure 5). Furthermore, we performed transcriptome sequencing on these two strains under high-temperature treatment. Comparative transcriptome analysis showed that compared to strain JY, strain HLM activated more DEGs under heat stress (Figure 6). This result also indicates that more DEGs in strain HLM are related to heat stress. Strain JY had more downregulated DEGs than strain HLM (Figure 6), also suggesting that strain HLM may possess a more complex gene regulatory network to cope with heat stress.

GO and KEGG enrichment analyses have been used as routine methods to analyze the transcriptomes of many edible fungi, such as Lentinula edodes [42,43], Stropharia rugosoannulata [44], and Pleurotus ostreatus [45]. Our results showed that after heat stress, the GO functions of strain HLM were mainly enriched in transporter activity, while those of strain JY were mainly enriched in catalytic activity. Previous studies on yeast and wheat resistance to high-temperature stress have demonstrated that DEGs are involved in transporter, catalytic, and oxidoreductase activities [46,47,48]. These processes are similar to those involved in Morchella after heat stress and may be necessary for its response to heat stress. Transporters are an important class of proteins [46,49], play roles in peptide transport, biological membrane formation, and fatty acid synthesis. They can protect cells from heat stress damage by enhancing membrane stability [50]. After heat stress in strain HLM, KEGG pathways were mainly enriched in ABC transporters, biotin metabolism, fatty acid biosynthesis, and fatty acid metabolism. In contrast, after heat stress in strain JY, KEGG was mainly enriched in glutathione metabolism, which was significantly downregulated. This result indicates that heat stress has an important impact on the metabolic activities of Morchella, especially the upregulation of transporter gene expression, suggesting that Morchella may improve its heat tolerance through transporters, biotin metabolism, and fatty acid biosynthesis.

Heat shock proteins (HSPs) are a class of important proteins that resist high temperature and other stresses [51,52]. They have the role of molecular chaperones to prevent protein misfolding and denaturation, and reduce the damage caused by stress [53]. Heat shock protein 70(HSP70) is the most important group of cell functional proteins in the heat shock protein family [54]. When subjected to stress such as high temperature, the transcriptional expression of its genes increases, which makes the organism’s function recoverable when it is under certain pressure [55]. The Yhsp70 gene has been cloned in corn, Eupatorium adenophorum and Eryngium adenophorum, and its expression increased under high-temperature stress [56]. Heat shock protein 20 (HSP20) is a small heat shock protein that plays an important role in plant growth, development, and stress resistance [57]. Under high-temperature stress, the expression of the HSP20 gene during maize growth and development is significantly upregulated [58]. In this study, the expression levels of genes encoding heat shock proteins HSP70 and HSP20 were significantly upregulated in the heat-tolerant strain but significantly downregulated in the heat-sensitive strain. The results indicate that the heat-tolerant strain can reduce damage caused by high temperature through the action of heat shock proteins, improving the strain’s heat tolerance and playing an important role in the resistance of mycelia to heat stress. This finding is consistent with the fact that heat shock protein genes are positively correlated with heat tolerance and enhance tolerance to salt, water, and high temperature in plants and fungi [59].

Aldo-keto reductases (AKRs) are a superfamily of enzymes that play crucial roles in various cellular processes, including the metabolism of xenobiotics, steroids, and carbohydrates [60]. They are often associated with the metabolism of exogenous and endogenous toxic substances, including those stimulated by stress [61]. AKR4C is the most studied in the AKR family in plants and is related to environmental stress tolerance [62,63,64]. Heat stress not only directly disrupts protein folding but also induces severe oxidative stress, leading to membrane lipid peroxidation and the accumulation of highly reactive carbonyl compounds such as malondialdehyde [65]. These toxic molecules can cross-link with proteins and nucleic acids, causing extensive secondary damage. AKR4C can reduce already formed carbonyl toxins like MDA by consuming NADPH [61], converting them into inert alcohols, thereby interrupting the vicious cycle of oxidative damage, preventing the accumulation of toxic products, and creating a favorable metabolic environment for cell repair and survival. In this study, the expression level of the AKR4C gene was upregulated in the heat-tolerant strain but downregulated in the heat-sensitive strain under high-temperature stress. Simultaneously, under high-temperature stress, the MDA content in the mycelia of the heat-tolerant strain did not change significantly, while it increased significantly in the heat-sensitive strain, which is consistent with the gene expression results. This indicates that the heat-tolerant strain has stronger carbonyl toxin clearance capability and resistance to high-temperature stress.

Glutathione (GSH) can scavenge harmful oxygen free radicals produced within cells and alleviate oxidative stress-induced damage [66]. High expression of the glutathione S-transferase (GST) gene can reduce oxidative stress and enhance heat tolerance [67]. In L. edodes strains, GSH expression was upregulated after heat stress and was used to eliminate superoxide damage to cell membranes [68]. Increased activities of peroxidase (POD) and catalase (CAT) can reduce oxidative damage caused by high temperature [69,70], and the expression of the catalase gene can improve the resistance of P. ostreatus to heat stress [71]. Pyruvate decarboxylase (PDC) plays an important role in fermentation metabolism pathways, participates in plant energy and substance metabolism, and responds to various biotic and abiotic stresses such as hypoxia, low temperature, and high salt [72]. In this study, after heat stress, the expression level of the GST gene was upregulated in the heat-tolerant strain but downregulated in the heat-sensitive strain. The expression levels of genes related to PDC, CAT, and POD were upregulated under high-temperature stress, and the upregulation amplitude was higher in the heat-tolerant strain than in the heat-sensitive strain. This suggests that the upregulated expression of these genes promotes the synthesis of their encoded products and enhances the strain’s ability to resist high-temperature stress through mutual collaboration. At the same time, under high-temperature stress, the activities of CAT and POD in the mycelia of the heat-tolerant strain were higher than those in the heat-sensitive strain, which is consistent with the gene expression results, indicating that the heat-tolerant strain has a stronger reactive oxygen species scavenging ability. This is consistent with the findings of Ren et al. (2021) on Stropharia rugosoannulata [73].

Tocopherols are lipid-soluble molecules that belong to the group of vitamin E compounds [74]. It participates in physiological processes such as germination and export of photoassimilates, and growth, leaf senescence, and plant responses to abiotic stresses [75]. 4-hydroxyphenylpyruvate dioxygenase (HPPD) is a key enzyme in aromatic amino acid metabolism, responsible for catalyzing the synthesis of tocopherols (vitamin E) [75]. Its activity is closely linked to the level of abiotic stress experienced by plants [76]. Previous studies have shown that HPPD can balance photosynthetic efficiency with repair of oxidative damage in Arabidopsis [77]. Overexpression of the HPPD gene can directly increase tocopherol content in organisms and enhance their tolerance to oxidative stress [78,79,80]. In this study, the expression level of the HPPD gene was upregulated in the heat-tolerant strain but downregulated in the heat-sensitive strain under high-temperature stress. This indicates that cells may have enhanced the biosynthesis of the lipid-soluble antioxidant molecule tocopherol. As the most direct antioxidant in the membrane system, tocopherol can efficiently quench singlet oxygen and interrupt lipid peroxidation chain reactions [75]. Therefore, the induction of HPPD in the heat-tolerant strain may strengthen the membrane system’s own defense by enhancing the biosynthesis of endogenous antioxidants (tocopherols). This works synergistically with the defense network constituted by water-soluble antioxidant enzyme systems (e.g., POD and CAT), the HSPs family, and the AKR detoxification system to collectively combat damage caused by heat stress.

Glucose-6-phosphate dehydrogenase (G6PDH) is a key enzyme in the pentose phosphate pathway, the enzyme catalyzes the first reaction in the pathway leading to the production of pentose phosphates and reducing power in the form of NADPH for reductive biosynthesis and maintenance of the redox state of the cell [81]. Moreover, the oxidation of pentose phosphate by G6PDH is one of the main pathways for the production of NADPH [82]. In this study, the expression level of the G6PDH gene was upregulated in the heat-tolerant strain but showed no significant change in the heat-sensitive strain under high-temperature stress. The high expression of G6PDH enhances the metabolic flux through the pentose phosphate pathway, substantially increasing the production level of the key intracellular reducing power, NADPH [83]. The produced NADPH can directly drive detoxification enzymes like AKR4C to reductively clear toxic carbonyl compounds. It can also provide power for GST-mediated conjugate detoxification by maintaining the reduced state of the glutathione and ascorbate pools, thereby collectively defending against the hazards brought by heat stress [84].

Sphingolipid is a component of the plasma membrane of eukaryotic cells [85]. SMPD1 is an important enzyme in sphingomyelin and ceramide metabolic pathways, and plays an important role in signal transmission [86]. Activation of acid sphingomyelinase (Asm) is crucial for the assembly and activation of the NADPH oxidase complex, which subsequently produces reactive oxygen species (ROS) [87]. In this study, the expression level of the SMPD1 gene was upregulated in the heat-tolerant strain but showed no significant change in the heat-sensitive strain under high-temperature stress, which is consistent with its increased expression in animal cells under stress [87]. Furthermore, related genes G6PDH and AKR4C were both upregulated. The purpose of SMPD1 upregulation might be to generate second messengers like ceramide, serving as a defense mobilization signal. Through downstream signaling, it promotes G6PDH expression [87,88]. After the upregulation of the G6PDH gene, the large amount of NADPH produced provides energy for all subsequent detoxification processes dependent on reducing power [83,84]. Finally, the upregulation of the AKR4C gene, as the terminal detoxification enzyme, directly utilizes NADPH to clear oxidative damage end-products (e.g., MDA) [60] (Figure 10). Therefore, the upregulation of the SMPD1 gene may be a key regulatory step for the strain to effectively convert stress signals into powerful protective outputs.

5. Conclusions

This study systematically evaluated the heat tolerance of 19 Morchella strains through combined analysis of phenotype, physiology, and transcriptomics, screening out the heat-tolerant strain HLM and the heat-sensitive strain JY. This research elucidates the complex, multi-layered defense network in the heat-tolerant Morchella strain HLM that achieves its exceptional thermotolerance. We propose a novel regulatory axis centered on sphingolipid-mediated signaling, complementing and reinforcing the classical heat stress response through a “stress perception—metabolic preparation—terminal detoxification” pathway. Upon heat shock, the rapid upregulation of SMPD1 may promote the generation of ceramide-based second messengers. These lipid signals, acting as defense alarms, likely coordinate adaptive metabolic shifts by activating specific kinase cascades to promote G6PDH expression, thereby driving carbon flux through the pentose phosphate pathway to produce a large amount of the universal reducing power currency—NADPH. Finally, AKR4C is induced as the terminal detoxification enzyme, directly utilizing this NADPH to specifically reduce toxic carbonyl end-products generated from lipid peroxidation, such as malondialdehyde, thus completing a full closed loop from damage signal to toxin clearance.

This novel sphingolipid signaling regulation mechanism operates synergistically with the classical heat stress defense system, forming a multi-dimensional defense strategy. In contrast to the upregulated molecular chaperones HSP70 and HSP20, which are primarily responsible for repairing denatured proteins and maintaining protein structural stability, the AKR4C- and GST-mediated detoxification systems focus on eliminating chemical toxins that attack protein functional groups: AKR4C clears reactive carbonyls (e.g., MDA) via direct reduction, while GST handles electrophilic toxins through glutathione conjugation. Together with HSPs, they guard the integrity of the proteome from two dimensions: “chemical toxicity clearance” and “physical structure repair.” Furthermore, the upregulation of genes related to PDC indicates adaptive adjustments in basal energy metabolism, which, together with the metabolic remodeling by G6PDH, optimizes resource allocation under stress. The core reducing power (NADPH) produced by G6PDH not only directly drives the reductive detoxification by AKR4C but also indirectly supports conjugate detoxification by GST through regenerating glutathione (GSH), while simultaneously providing the necessary electron donors for the continuous operation of upregulated antioxidant enzymes like CAT and POD. This finding reveals a new regulatory paradigm in fungi for achieving systemic thermotolerance beyond traditional heat shock responses, through the integration of signal transduction, metabolic reprogramming, protein protection, redox homeostasis, and chemical detoxification networks. These findings provide new insights into the heat response mechanisms of large ascomycete fungi and offer valuable genetic resources for breeding heat-tolerant Morchella varieties. This study also lays a theoretical foundation for promoting cultivation in warmer regions and provides support for further research into the heat tolerance mechanisms of other edible fungi.

Simultaneously, we acknowledge that the regulatory model proposed in this study is derived from an in-depth analysis of strains with extreme phenotypes. Its general applicability requires further validation across a broader spectrum of Morchella germplasm resources and other edible fungi. Therefore, validating this pathway within a wider genetic context and exploring its interactions with species-specific regulatory networks will be an important direction for future research.